1. Field of the Invention
This invention concerns the enzyme acetylcholinesterase (AChE) in which the inventors have identified a biologically active peptide.
2. Description of the Related
The classical or cholinergic role of AChE is to degrade enzymatically extracellular acetylcholine. However, it has long been known that AChE exists also in a soluble form, (not a requirement for its classic enzymatic role) and is found in parts of the body where there is little or no acetylcholine. It is becoming widely accepted that AChE has a non-cholinergic function, though the biochemical basis for this function remains unclear.
It is believed that excessive AChE may enhance calcium entry into cells independent of its normal enzymatic action. Elevated cellular calcium levels may lead to a range of pernicious consequences, including undesirable changes in gene expression and, more importantly, mitochondrial swelling which may thereby compromise ATP metabolism and may indeed lead to apoptosis or programmed cell-death. Disease states which may be implicated include Parkinson's disease, Alzheimer's disease, stroke and malignancy.
The cytoplasm of cells typically contains calcium at concentrations of the order of 1 μm. Calcium is present intracellularly in the endoplasmic reticulum in millimolar concentrations. Extracellular body fluids contain calcium also in millimolar concentrations. A calcium pump operates to maintain this substantial concentration difference between the cytoplasm and the endoplasmic reticulum, and thapsigargin is known to be implicated in the breakdown of this pump. Similarly a calcium pump normally functions between the cytoplasm and the extracellular fluid. It is believed that the consequences of the action of excessive AChE may be comparable to the breakdown of these pumps.
AChE, acting in a non-cholinergic capacity, has been shown to play an important part in the normal and abnormal functioning of the substantia nigra, the region affected in Parkinson's disease. There are are three possible ways in which AChE may have toxic effects:    (i) excessive AChE may be released as a consequence of compensatory mechanisms known to occur in that disorder;    (ii) excessive glutamatergic activity known to occur in Parkinson's disease may lead to over-stimulation of calcium channel N-methyl-D-aspartate (NMDA) glutamate receptors, thereby converting a physiological situation to a pathological one;    (iii) normal levels of AChE may act synergistically with fragments of β-amyloid precursor proteins known to be present in the Parkinsonian substantia nigra.
AChE, again acting in a non-cholinergic capacity, may be an important contributing factor in Alzheimer's disease. In transgenic mice with excessive AChE there are cognitive deficits reminiscent of Alzheimer's disease. Moreover Alzheimer's disease has been directly associated with inappropriate levels and forms of AChE. Excessive AChE may act to enhance calcium entry through overactivation of otherwise normal adaptive processes via a mechanism discussed in the experimental section below.
Current therapies for both degenerative diseases are somewhat inadequate. Anti-Parkinsonian drugs which target dopamine substitution do not arrest neuronal cell loss, and newer drugs aiming to block calcium entry directly may have poor net payoff in terms of neuronal health and in addition would have widespread undesirable effects in both the central nervous system and peripheral tissue. Moreover, drugs used in Alzheimer's disease which exclusively target the cholinergic system, neglect areas where AChE may be having its pivotal non-cholinergic function. Previous attempts to target calcium channel activity in therapy for neurodegenerative disorders have been hampered by the non-selective effects of the compounds available.
In order to be conveniently administered, a compound for treatment of disorders of the central nervous system, or more particularly of the brain, needs to be capable of crossing the blood-brain barrier. AChE is not capable of doing this, though a small lipid-soluble analogue of part of this molecule might be. Workers in the field have been seeking biologically active peptides based on the AChE molecule for more than ten years, in the hope of thereby achieving a more effective and selective treatment for disorders of the central nervous system such as Alzheimer's and Parkinson's diseases.
It is known that antagonism of NMDA receptors is being explored as a therapy for stroke. The present invention is expected to find application in specific therapies for combating stroke and other problems of cerebral circulation.
Abnormal cholinesterase expression occurs in several types of tumour cells. Although the role of cholinesterases in tumorigenesis is unclear, the fact that AChE and BuChE (butyryl cholinesterase) may be involved in the control of cell growth and proliferation during early development suggests that the amplification of cholinesterase genes may influence the ability of tumour cells to proliferate more rapidly. According to the invention, antagonists of the non-cholinergic action of AChE are expected to be of interest in the prophylaxis and treatment of cancer.
Several separate lines of evidence suggest that motor neurones may share, along with the neurones that are lost in Parkinson's disease (substantial nigra) and in Alzheimer's disease (basal forebrain, locus couruleus, raphe nucleus) several distinctive features as well as the common characteristic of releasing AChE in a non-cholinergic capacity. The released AChE may have a novel action, as in the regions prone to Parkinsonian or Alzheimer degeneration to enhance developmental mechanisms in immature populations of motor neurones but exert toxic actions if inappropriately reactivated in mature systems. The AChE-peptide described herein may also therefore be pivotal in the aetiology of Motor Neurone Disease. The undisclosed finding supporting this claim is that in pilot studies, the AChE peptide binds bilaterally to selective sites within the spinal cord.
Amyloid precursor protein (APP) is known to have similar features to AChE as follows. Both AChE and APP are secreted from neurons into the cerebro spinal fluid (CSF), where for both AChE and APP there is a decrease in CSF levels in Alzheimer's disease. Both AChE and APP can have trophic functions.
Both AChE and β-amyloid enhance calcium entry through NMDA receptors. Both AChE and APP activate potassium channels, probably linked to changes in intracellular calcium. Both AChE and β-amyloid activate macrophages. Low stimulation of NMDA receptors has trophic effects whereas high stimulation is toxic. The dual trophic-toxic action of both APP and AChE may thus be mediated via NMDA receptors. A similar dual action via NMDA receptors has already been shown for the trophic factor BDNF in cortical cells. Finally, β-amyloid and the monomer of AChE can bind together as a complex.
This invention results from the inventors' identification of a region of the AChE molecule from which a biologically active peptide (obtained either synthetically or by endogenous processing) can be derived. The peptide consists of 14 residues of AChE from residue 535 to residue 548 of the mature protein (in the translation of the mRNA sequence, EMBL accession hsache. empri, number M55040, beginning at nucleotide 310). The sequence of this peptide is amino Ala-Glu-Phe-His -Arg-Trp-Ser-Ser-Tyr-Met-Val-His-Trp-Lys-carboxy (SEQ. ID No: 1), or in the one letter code, AEFHRWSSYMVHWK. The inventors propose that this, or a related, peptide from this region of AChE acts alone or in synergism with a fragment of beta-amyloid to contribute to neuronal degeneration. The invention thus provides in one aspect a peptide containing at least six amino acid residues and having at least 70% homology with part or all of the above sequence. Preferably the peptide contains at least 12 amino acid residues having at least 90% homology with the above sequence.